Caspases and apoptosis

ABSTRACT

A compound of formula                    
     wherein 
     R 1  is hydrogen, or C 1-4  alkyl; 
     R 2  is C 1-10  alkyl, optionally substituted arylC 1-4 alkyl, optionally substituted heteroaryl C 1-4  alkyl optionally substituted C 3-7  cycloalkyl, or R 1  and R 2  together with the nitrogen to which they are attached from a 3 to 10 membered ring which optionally contains an aditional heteroatom selected from oxygen, nitrogen or sulfur, 
     R 3  and R 4  are C 1-6 alkyl, hydrogen, nitro, or halogen and 
     R 5  is C 1-6 alkyl, hydrogen, arylalkyl or heteroarylalkyl. 
     The present invention is to the novel compounds of Formula (I), their pharmaceutical compositions, and to the novel inhibition of Caspases for use in the treatment of apoptosis, and disease states caused by excessive or inappropriate cell death.

This application is a 371 of PCT/US98/15909 Jul. 30, 1998, which claimsbenefit of Ser. No. 60/054,249 Jul. 30, 1997.

FIELD OF THE INVENTION

The present invention is to the discovery of a new method to blockexcessive or inappropriate apoptosis in a mammal.

BACKGROUND

It has been recognized for over a century that there are different formsof cell death. One form of cell death, necrosis, is usually the resultof severe trauma and is a process that involves loss of membraneintegrity and uncontrolled release of cellular contents, often givingrise to inflammatory responses. In contrast, apoptosis is a morephysiological process that occurs in a controlled manner and isgenerally non-inflammatory in nature. For this reason apoptosis is oftenreferred to as programmed cell death. The name itself (apoptosis: Greekfor “dropping off”, for example leaves from trees) implies a cell deaththat is part of a normal physiological process (Kerr et al., Br. J.Cancer, 26: 239-257 (1972)).

Apoptosis appears to be a carefully controlled series of cellular eventswhich ultimately leads to death of the cell. This process forelimination of unwanted cells is active and requires expenditure ofcellular energy. The morphological characteristics of apoptosis includecell shrinkage and loss of cell-cell contact, condensation of nuclearchromatin followed by fragmentation, the appearance of membraneruffling, membrane blebbing and apoptotic bodies. At the end of theprocess, neighboring cells and macrophages phagocytose the fragmentsfrom the apoptotic cell. The process can be very fast, occurring in aslittle as a few hours (Bright et al., Biosci. Rep., 14: 67-82 (1994)).

The best defined biochemical event of apoptosis involves the orderlydestruction of nuclear DNA. Signals for apoptosis promote the activationof specific calcium- and magnesium-dependent endonucleoases that cleavethe double stranded DNA at linker regions between nucleosomes. Thisresults in production of DNA fragments that are multiples of 180-200base pair fragments (Bergamaschi et al., Haematologica, 79: 86-93(1994); Stewart, JNCI, 86: 1286-1296 (1994)). When examined by agarosegel electrophoresis, these multiple fragments form a ladder pattern thatis characteristic for most cells undergoing apoptosis.

There are numerous stimuli that can signal cells to initiate or promotecellular apoptosis, and these can be different in different cells. Thesestimuli can include glucocorticoids, TNFa, growth factor deprivation,some viral proteins, radiation and anticancer drugs. Some of thesestimuli can induce their signals through a variety of cell surfacereceptors, such as the TNF/nerve growth factor family of receptors,which include CD40 and Fas/Apo-1 (Bright et al., supra). Given thisdiversity in stimuli that cause apoptosis it has been difficult to mapout the signal transduction pathways and molecular factors involved inapoptosis. However, there is evidence for specific molecules beinginvolved in apoptosis.

The best evidence for specific molecules that are essential forapoptosis comes from the study of the nematode C. elegans. In thissystem, genes that appear to be required for induction of apoptosis areCed-3 and Ced-4. These genes must function in the dying cells and, ifeither gene is inactivated by mutation, cell death fails to occur (Yuanet al., Devel. Biol., 138: 33-41 (1990)). In mammals, genes that havebeen linked with induction of apoptosis include the proto-oncogene c-mycand the tumor suppresser gene p53 (Bright et al., supra; Symonds et al.,Cell, 78: 703-711 (1994)).

In this critical determination of whether or not to undergo apoptosis,it is not surprising that these are genes that program for proteins thatinhibit apoptosis. An example in C. elegans is Ced-9. When it isabnormally activated, cells survive that would normally die and,conversely, when Ced-9 is inactivated cells die that would normally live(Stewart, B. W., supra). A mammalian counterpart is bcl-2, which hadbeen identified as a cancer-causing oncogene. This gene inhibitsapoptosis when its product is overexpressed in a variety of mammaliancells, rendering them less sensitive to radiation, cytotoxic drugs andapoptotic signals such as c-myc (Bright et al., supra). Some virusprotein have taken advantage of this ability of specific proteins toblock apoptosis by producing homologous viral proteins with analogousfunctions. An example of such a situation is a protein produced by theEpstein Barr virus that is similar to bcl-2, which prevents cell deathand thus enhances viral production (Wells et al., J. Reprod. Fertil.,101: 385-391 (1994)). In contrast, some proteins may bind to and inhibitthe function of bcl-2 protein, an example being the protein bax(Stewart. B. W., supra). The overall picture that has developed is thatentry into apoptosis is regulated by a careful balancing act betweenspecific gene products that promote or inhibit apoptosis (Barinaga,Science, 263: 754-756 (1994).

Apoptosis is an important part of normal physiology. The two most oftensited examples of this are fetal development and immune celldevelopment. In development of the fetal nervous system, over half ofthe neurons that exist in the early fetus are lost by apoptosis duringdevelopment to form the mature brain (Bergamaschi et al., Haematologica,79: 86-93 (1994)). In the production of immune competent T cells (and toa lesser extent evidence exists for B cells), a selection process occursthat eliminates cells that recognize and react against self. Thisselection process is thought to occur in an apoptotic manner withinareas of immune cell maturation (Williams, G. T., J. Pathol., 173: 1-4(1994); Krammer et al., Curr. Opin. Immunol., 6: 279-289 (1994)).

Dysregulation of apoptosis can play an important role in disease states,and diseases can be caused by both excessive or too little apoptosisoccurring. An example of diseases associated with too little apoptosiswould be certain cancers. There is a follicular B-cell lymphomaassociated with an aberrant expression of functional bcl-2 and aninhibition of apoptosis in that cell (Bergamaschi et al., supra). Thereare numerous reports that associate deletion or mutation of p53 with theinhibition of apoptosis and the production of cancerous cells (Kerr etal., Cancer, 73: 2013-2026 (1994); Ashwell et al., Immunol. Today, 15:147-151, (1994)). In contrast, one example of excessive or inappropriateapoptosis is the loss of neuronal cells that occurs in Alzheimerdisease, possible induced by b-amyloid peptides (Barr et al.,BioTechnology, 12: 487-493 (1994)). Other examples include excessiveapoptosis of CD4⁺ T cells that occurs in HIV infection, of cardiacmyocytes during infarction/reperfusion and of neuronal cells duringischemia (Bergamaschi et al., supra); Barr et al., supra).

Some pharmacological agents attempt to counteract the lack of apoptosisthat is observed in cancers. Examples include topoisomerase IIinhibitors, such as the epipodophyllotoxins, and antimetabolites, suchas ara-c, which have been reported to enhance apoptosis in cancer cells(Ashwell et al., supra). In many cases with these anti-cancer drugs, theexact mechanism for the induction of apoptosis remains to be elucidated.

In the last few years, evidence has built that ICE and proteinshomologous to ICE (Caspases) play a key role in apoptosis. This area ofresearch has been spurred by the observation of homology between theprotein coded by Ced-3, a gene known to be critical for C. Elegansapoptosis, and ICE (Caspase 1). These two proteins share 29% amino acididentity, and complete identity in the 5 amino acid portion thought tobe responsible for protease activity (QACRG) (Yuan et al., Cell, 75:641-652 (1993)). Additional homologies are observed between ICE and theproduct of the nedd-2 gene in mice, a gene suspected of involvement inapoptosis in the developing brain (Kumar et al., Genes Dev., 8:1613-1626 (1994)) and Ich-1 (Caspase 2) and CPP32 (Caspase 3), humancounterparts of nedd-2 isolated from human brain cDNA libraries (Wang etal., Cell, 78: 739-750 (1994); Fernandes-Alnemiri et al., J. Biol.Chem., 269: 30761-30764(1994)).

Further proof for the role of these proteins in apoptosis comes fromtransfection studies. Over expression of murine ICE caused fibroblaststo undergo programmed cell death in a transient transfection assay(Miura et al., Cell, 75: 653-660 (1993)). Cell death could be preventedby point mutations in the transfected gene in the region of greatesthomology between ICE and Ced-3. As very strong support for the role ofICE in apoptosis, the authors showed that ICE transfection-inducedapoptosis could be antagonized by overexpression of bcl-2, the mammalianoncogene that can prevent programmed cell death (Miura et al., supra).Additional experiments were performed using the crmA gene. This gene ofthe cowpox virus encodes a serpin protein, a family of proteins that areinhibitors of proteases (Ray et al., Cell, 69: 597-604 (1992)).Specifically, the protein of crmA has been shown to inhibit processingof pro-interleukin-1b by ICE. (Gagliardini et al. Science, 263: 826-828(1994)) showed that microinjection of the crmA gene into dorsal rootganglion neurons prevented cell death induced by nerve growth factordeprivation. This result shows that ICE is involved in neuronal cellapoptosis. A more direct demonstration of ICE involvement comes fromexperiments in which ICE transfection is coupled with the co-expressionof crmA, demonstrating a crmA-induced suppression of the ICE-inducedapoptosis response (Miura et al., supra; Wang et al., supra).

In addition to ICE, researchers have examined the ability of Caspases topromote apoptosis. (Kumar et al. supra) demonstrated that overexpression of nedd-2 in fibroblasts and neuroblastoma cells resulted incell death by apoptosis and that this apoptosis could also be suppressedby expression of the bcl-2 gene. Most recently. Wang et al., (Wang etal., supra) examined the over expression of Ich-1 in a number ofmammalian cells. Expression resulted in cell apoptosis, which could beantagonized by bcl-2 co-expression. Mutation of a cysteine residue,contained within the QACRG motif and presumed to be critical forprotease function, to serine abolished apoptotic activity.

Further evidence for a role of a cysteine protease in apoptosis comesfrom a recent report by Lazebnik et al. (Nature, 371: 346-347 (1994)).These authors have used a cell-free system to mimic and study apoptosis.In their system there is a protease activity that cleaves the enzymepoly(ADP-ribose) polymerase at a site identical to a cleavage site inpre-interleukin-1b. However, this yet to be isolated protease and ICEappear to be different and to act on different substrate proteins.Blockade of protease activity in the system, using non-selectivecysteine protease inhibitors, resulted in inhibition of apoptosis.

Taken together, the above evidence provides striking involvement ofCaspases in the induction of apoptosis in mammalian cells. Braininterleukin-1 has been reported to be elevated in Alzheimer disease andDown syndrome (Griffin et al., Proc. Natl. Acad. Sci. U.S.A., 86:7611-7615 (1989)). There are also reports that interleukin-1 canincrease the mRNA and production of b-amyloid protein, a major componentof senile plaques in Alzheimer disease as well as in brains of peoplewith Down syndrome and with aging (Forloni et al., Mol. Brain Res., 16:128-134 (1992); Buxbaum et al., Proc. Natl. Acad. Sci. U.S.A., 89:10075-10078 (1992); Goldgaber et al., Proc. Natl. Acad. Sci. U.S.A., 86:7606-7610 (1989)). These reports can be viewed as additional evidencefor the involvement of ICE in these diseases and the need for use of anovel therapeutic agent and therapy thereby.

To date, no useful therapeutic strategies have blocked excessive orinappropriate apoptosis. In one patent application, EPO 0 533 226 anovel peptide structure is disclosed which is said to be useful fordetermining the activity of ICE, and therefore useful in the diagnosesand monitoring of IL-1 mediated diseases. Therefore, a need exists tofind better therapeutic agents which have non-toxic pharmacological andtoxicological profiles for use in mammals. These compounds should blockexcessive or inappropriate apoptosis cells, and hence provide treatmentfor diseases and conditions in which this condition appears.

SUMMARY OF THE INVENTION

The present invention is to the novel compounds of Formula (I), theirpharmaceutical compositions, and to the novel inhibition of Caspases foruse in the treatment of apoptosis, and disease states caused byexcessive or inappropriate cell death. The compounds of Formula I aremost effective in inhibiting Caspases three and seven.

Another aspect of the present invention is to a pharmaceuticalcomposition comprising a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier ordiluent.

Another aspect of the present invention is to a method for the treatmentof diseases or disorders associated with excessive IL-1b convertaseactivity, in a mammal in need thereof, which method comprisesadministering to said mammal an effective amount of a compound ofFormula (I), or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention is to a method of preventing orreducing apoptosis in a mammal, preferably a human, in need of suchtreatment which method comprises administering to said mammal or humanan effective amount of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof.

Another aspect of the present invention is to a method of blocking ordecreasing the production of IL-1b and/or TNF, in a mammal, preferably ahuman, in need of such treatment which method comprises administering tosaid mammal or human an effective amount of a compound of Formula (I),or a pharmaceutically acceptable salt thereof.

The compounds of Formula I are represented by the structure

wherein

R₁ is hydrogen, or C₁₋₄ alkyl;

R₂ is C₁₋₁₀ alkyl, optionally substituted arylC₁₋₄alkyl, optionallysubstituted heteroaryl C₁₋₄ alkyl, optionally substituted C₃₋₇cycloalkyl, or R₁ and R₂ together with the nitrogen to which they areattached from a 3 to 10 membered ring which optionally contains anaditional heteroatom selected from oxygen, nitrogen or sulfur;

R₃ and R₄ are C₁₋₆alkyl, hydrogen, nitro, or halogen and

R₅ is C₁₋₆alkyl, hydrogen, arylalkyl or heteroarylalkyl.

Preferably R₁ and R₂ are joined for form a five membered nitrogencontaining ring. It is recognized that the alkyl group in the arylalkylor heteroalkyl moiety may be branched or straight, such as a methyleneor a substituted methylene group, i.e., —CH(CH₃)— aryl. The optionallysubstituted aryl moiety of the arylalkyl group, may be substituted oneto three times independently by hydroxy, halogen, alkyl or alkoxy. R₅ ispreferably benzyl.

Compounds exemplified by Formula (I) include, but are not limited to:

5-Chlorosulfonyl-3,3-dichloro-2-oxindole

5-Benzylaminosulfonyl-3,3-dichloro-2-oxindole

5-[N-(1-Methyl-3-phenylpropylamino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(N-Benzyl-2-cyanoethylamino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(2-(3-Pyridyl)ethylamino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(2-Furfurylamino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(2-Isopropoxyethylamino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(2-Methoxyethylamino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(2-Tetrahydrofurfurylamino)sulfonyl]-3,3-dichloro-2-oxindole

(−)-5-[N-(cis-Myrtanylamino)sulfonyl]-3,3-dichloro-2-oxindole

5-[(1-Benzylpiperidinyl-4-amino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(2-Indanamino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(Cyclopropylmethylamino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(1,5-Dimethylhexylamino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(N-Methylbenzylamino)sulfonyl]-3,3-dichloro-2-oxindole

(±)-5-[N-(3-(N-Acetyl-N-methylamino)pyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole

5-[2-(1,2,3,4-Tetrahydroisoquinolino)sulfonyl]-3,3-dichloro-2-oxindole

(±)-5-[1-(Decahydroisoquinolino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(N-Methyl-2-cyanoethylamino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(N-Methylcyanomethylamino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(Pyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(N-Methylphenethylamino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(Azacyclooctane)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(3-Azabicyclo[3.2.2]nonane)sulfonyl]-3,3-dichloro-2-oxindole

5-[1-(2-Ethoxycarbonylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(Morpholino)sulfonyl]-3,3-dichloro-2-oxindole

(S)-(+)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(N-Methyl-2-(4-pyridinyl)ethylamino)sulfonyl]-3,3-dichloro-2-oxindole

5-[N-(N-Methyl-2-hydroxyethylamino)sulfonyl]-3,3-dichloro-2-oxindole

(S)-(+)-5-[N-(2-Hydroxymethylpyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole

(±)-5-[1-(3-Hydroxypyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole

(±)-5-[1-(3-aminocarbonylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

(±)-5-[1-(2-Methylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

(±)-5-[1-(4-Methylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

5-[1-(4-Hydroxypiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

(±)-5-[1-(2-(2-Hydroxyethyl)piperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

(±)-5-[1-(3-Hydroxymethylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

5-[1-(4-Phenylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

5-[1-(4-Benzylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

5-[1-(4-(1-Piperidinyl)piperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

5-Benzylaminosulfonyl-N-methyl-3,3-dichloro-2-oxindole

5-Chlorosulfonyl-N-methyl-3,3-dichloro-2-oxindole

5-Benzylaminosulfonyl-N-methyl-3,3-dichloro-2-oxindole

N-Methyl-5-(1-piperidinylsulfonyl)-3,3-dichloro-2-oxindole

The term “excessive IL-1b convertase activity” is used herein to mean anexcessive expression of the protein, or activation of the enzyme.

The term “C₁₋₆ alkyl” or “alkyl” is used herein to mean both straightand branched chain radicals of 1 to 6 carbon atoms, unless the chainlength is otherwise specified, including, but not limited to, methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl,and the like.

The term “heteroaryl” (on its own or in any combination, such as“heteroaryloxy”, or “heteroaryl alkyl”) is used herein to mean a 5-10membered aromatic ring system in which one or more rings contain one ormore heteroatoms selected from the group consisting of N, O or S, suchas, but not limited, to pyrrole, pyrazole, furan, thiophene, quinoline,isoquinoline, quinazolinyl, pyridine, pyrimidine, oxazole, oxadiazole,tetrazole, thiazole, thiadiazole, triazole, imidazole, benzimidazole,benzothiaphene, benzopyrrole, or benzofuran.

The term “aryl” (on its own or in any combination, such as “aryloxy”, or“arylalkyl”) is used herein to mean a phenyl and naphthyl ring.

The term “cycloalkyl” is used herein to mean cyclic radicals, preferablyof 3 to 7 carbons, including but not limited to cyclopropyl,cyclopentyl, cyclohexyl, and the like.

The term “halo” or “halogens”, is used herein to include, unlessotherwise specified, chloro, fluoro, bromo and iodo.

The present invention contains the inhibition of Caspases by compoundsof Formula (I). What is meant by the term “Caspases” are fragment,homologs, analogs and derivatives of the polypeptides Interleukin-1bconverting enzyme (or convertase). These analogs are structurallyrelated to the Caspase family. They generally encode a protein(s) whichexhibits high homology to the human ICE over the entire sequence.Preferably, the pentapeptide QACRG is conserved. The Caspases, which mayinclude many natural allelic variants (such as substitutions, deletionor addition of nucleotides) does not substantially alter the function ofthe encoded polypeptide. That is they retain essentially the samebiological function or activity as the ICE protease, although it isrecognized that the biological function may be enhanced or reducedactivity. The suitable activity is not IL-1b convertase activity, butthe ability to induce apoptosis or involved in programmed cell death insome manner. Suitable Caspases encompasses within this invention arethose described in PCT US94/07127 filed Jun. 23, 1994; and in U.S. Ser.No. 08/334,251, filed Nov. 1, 1994, whose disclosures are incorporatedherein by reference in their entirety.

The term “blocking or inhibiting, or decreasing the production of IL-1band/or TNF” as used herein refers to:

a) a decrease of excessive levels, or a down regulation, of the cytokinein a human to normal or subnormal levels by inhibition of the in vivorelease of the cytokine; or

b) a down regulation, at the genomic level, of excessive in vivo levelsof the cytokine (IL-1 or TNF) in a human to normal or sub-normal levels;or

c) a down regulation, by inhibition of the direct synthesis of thecytokine (IL-1, or TNF) as a postranslational event; or

d) a down regulation, at the translational level, of excessive in vivolevels of the cytokine (IL-1, or TNF) in a human to normal or sub-normallevels.

The blocking or inhibiting, or decreasing the production of IL-1b and/orTNF is a discovery that the compounds of Formula (I) are inhibitors ofthe cytokines, IL-1 and TNF is based upon the effects of the compoundsof Formulas (1) on the production of the IL-1 and TNF in in vitro and invivo assays which are well known and recognized in the art, some ofwhich are described herein.

Compound of the present invention may be synthesized in accordance withthe schemes illustrated below.

5-Alkylaminosulfonyl-3,3-dichloro-2-oxindoles

Isatin or its N-alkyl derivative is treated with chlorosulfonic acid attemperatures ranging from 0-10° C. in order to obtain5-chlorosulfonyl-3,3-dichloro-2-oxindole, the direct precursor to thecompounds of this invention. Treatment of the chlorosulfonyl derivativewith a primary or secondary amine in organic solvents such astetrahydrofuran, methylene chloride or dimethylformamide with or withoutthe addition of a tertiary amine base such as triethylamine yields the5-alkylaminosulfonyl-3,3-dichloro-2-oxindole.

EXAMPLE 1 (SB263831) a) 5-Chlorosulfonyl-3,3-dichloro-2-oxindole

A solution of isatin (1.6 g, 10 mmol) in chlorosulfonic acid (6.6 mL)was heated to 70° C. for 3 h. After cooling to RT, the solution waspoured into ice and extracted with methylene chloride. The organicsolution was dried over magnesium sulfate, filtered, and the solvent wasremoved under reduced pressure to afford the title compound as an orangesolid in quantitative yield. ¹H NMR (400 MHz, CDCl₃) d7.25 (d, J=10.5Hz, 1H), 8.12 (d, J=10.5 Hz, 1H), 8.29 (s, 1H), 8.74 (br s, 1H).

b) 5-Benzylaminosulfonyl-3,3-dichloro-2-oxindole

To a solution of 5-chlorosulfonyl-3,3-dichloro-2-oxindole (100 mg, 400umol) in methylene chloride (3 mL) was added benzylamine (135 uL, 1.2mmol) dropwise. After stirring for 4 h. 3 N hydrochloric acid was addedalong with an additional volume of methylene chloride (20 mL). Theorganic layer was dried over magnesium sulfate, filtered, and silica gelflash chromatography (25 to 35% ethyl acetate/hexanes) yielded the titlecompound. ES (−) MS m/e=369 (M−H).

EXAMPLE 2 (SB264862)5-[N-(1-Methyl-3-phenylpropylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting1-methyl-3-phenylpropylamine for benzylamine afforded the title compoundas a yellow foam in 32% yield. ES (+) MS m/e=413 (M+H).

EXAMPLE 3 (SB264860)5-[N-(N-Benzyl-2-cyanoethylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substitutingN-(2-cyanoethyl)benzylamine for benzylamine afforded the title compoundas a white solid in 19% yield. ES (+) MS m/e=424 (M−H).

EXAMPLE 4 (SB265240)5-[N-(2-(3-Pyridyl)ethylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting2-(3-pyridyl)ethylamine for benzylamine afforded the title compound as ayellow oil in 4% yield. ES (+) MS m/e=386 (M+H).

EXAMPLE 5 (SB265241)5-[N-(2-Furfurylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting2-furfurylamine for benzylamine afforded the title compound as a yellowsolid in 17% yield. ES (−) MS m/e=359 (M−H).

EXAMPLE 6 (SB265242)5-[N-(2-Isopropoxyethylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting2-isopropoxyethylamine for benzylamine afforded the title compound as ayellow foam in 33% yield. ES (−) MS m/e=365 (M−H).

EXAMPLE 7 (SB265243)5-[N-(2-Methoxyethylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting2-methoxyethylamine for benzylamine afforded the title compound as ayellow foam in 27% yield. ES (−) MS m/e=337 (M−H).

EXAMPLE 8 (SB265244)5-[N-(2-Tetrahydrofurfurylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting2-tetrahydrofurfurylamine for benzylamine afforded the title compound asa light yellow foam in 30% yield. ES (−) MS m/e=363 (M−H).

EXAMPLE 9 (SB265246)(−)-5-[N-(cis-Myrtanylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting(−)-cis-myrtanylamine for benzylamine afforded the title compound as alight yellow foam in 8% yield. ES (−) MS m/e=415 (M−H).

EXAMPLE 10 (SB265247)5-[(1-Benzylpiperidinyl-4-amino)aminosulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting4-amino-1-benzylpeiperdine for benzylamine afforded the title compoundas a yellow oil in 5% yield. ES (−) MS m/e=452 (M−H).

EXAMPLE 11 (SB265248)5-[N-(2-Indanamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting2-indanamine for benzylamine afforded the title compound as a brown foamin 50% yield. ES (−) MS m/e=395 (M−H).

EXAMPLE 12 (SB265249)5-[N-(Cyclopropylmethylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substitutingcyclopropylmethylamine for benzylamine afforded the title compound as ayellow foam in 21% yield. ES (−) MS m/e=333 (M−H).

EXAMPLE 13 (SB265250)5-[N-(1,5-Dimethylhexylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting1,5-dimethylhexylamine for benzylamine afforded the title compound as ayellow foam in 12% yield. ES (−) MS m/e=391 (M−H).

EXAMPLE 14 (SB265550)5-[N-(N-Methylbenzylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substitutingN-methylbenzylamine for benzylamine afforded the title compound as ayellow solid in 38% yield. ES (−) MS m/e=384 (M−H).

EXAMPLE 15 (SB265551)(±)-5-[N-(3-(N-Acetyl-N-methylamino)pyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting3-(N-acetyl-N-methylamino)pyrrolidine for benzylamine afforded the titlecompound as a yellow foam in 39% yield. ES (−) MS m/e=404 (M−H).

EXAMPLE 16 (SB265594)5-[2-(1,2,3,4-Tetrahydroisoquinolino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting1,2,3,4-tetrahydroisoquinoline for benzylamine afforded the titlecompound as a yellow solid in 47% yield. ES (−) MS m/e=395 (M−H).

EXAMPLE 17 (SB265595)(±)-5-[1-(Decahydroisoquinolino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substitutingdecahydroisoquinoline for benzylamine afforded the title compound as ayellow foam in 27% yield. ES (−) MS m/e=401 (M−H).

EXAMPLE 18 (SB265596)5-[N-(N-Methyl-2-cyanoethylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substitutingN-methyl-beta-alaninenitrile for benzylamine afforded the title compoundas a light yellow solid in 28% yield. ES (−) MS m/e=346 (M−H).

EXAMPLE 19 (SB265597)5-[N-(N-Methylcyanomethylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substitutingN-methylaminoacetonitrile for benzylamine afforded the title compound asa yellow solid in 7% yield. ES (−) MS m/e=332 (M−H).

EXAMPLE 20 (SB265598)5-[N-(Pyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substitutingpyrrolidine for benzylamine afforded the title compound as a lightyellow solid in 21% yield. ES (−) MS m/e=333 (M−H).

EXAMPLE 21 (SB265599)5-[N-(N-Methylphenethylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substitutingN-Methylphenethylamine for benzylamine afforded the title compound as alight yellow foam in 45% yield. ES (−) MS m/e=397 (M−H).

EXAMPLE 22 (SB265600)5-[N-(Azacyclooctane)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substitutingazacyclooctane for benzylamine afforded the title compound as a yellowfoam in 49% yield. ES (−) MS m/e=375 (M−H).

EXAMPLE 23 (SB265601)5-[N-(3-Azabicyclo[3.2.2]nonane)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting3-azabicyclo[3.2.2]nonane for benzylamine afforded the title compound asa yellow foam in 60% yield. ES (−) MS m/e=387 (M−H).

EXAMPLE 24 (SB265602)5-[1-(2-Ethoxycarbonylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substitutingethyl piperidine-2-carboxylate for benzylamine afforded the titlecompound as a light yellow foam in 69% yield. ES (−) MS m/e=419 (M−H).

EXAMPLE 25 (SB265603) 5-[N-(Morpholino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substitutingmorpholine for benzylamine afforded the title compound as a light yellowsolid in 56% yield. ES (−) MS m/e=349 (M−H).

EXAMPLE 26 (SB265604)(S)-(+)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting(S)-(+)-2-(methoxymethyl)pyrrolidine for benzylamine afforded the titlecompound as a light yellow foam in 38% Yield. ES (−) MS m/e=377 (M−H).

EXAMPLE 27 (SB265605)5-[N-(N-Methyl-2-(4-pyridine)ethylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substitutingN-methyl-2-(4-pyridine)ethylamine for benzylamine afforded the titlecompound as a brown solid in 38% yield. ES (−) MS m/e=398 (M−H).

EXAMPLE 28 (SB265606)5-[N-(N-Methyl-2-hydroxyethylamino)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substitutingN-(methyl)aminoethanol for benzylamine afforded the title compound as alight yellow foam in 27% yield. ES (−) MS m/e=337 (M−H).

EXAMPLE 29 (SB265607)(S)-(+)-5-[N-(2-Hydroxymethylpyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting(S)-(+)-2-(hydroxymethyl)pyrrolidine for benzylamine afforded the titlecompound as a light yellow foam in 31% yield. ES (−) MS m/e=363 (M−H).

EXAMPLE 30 (SB265608)(±)-5-[1-(3-Hydroxypyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting3-hydroxypyrrolidine for benzylamine afforded the title compound as awhite solid in 9% yield. ES (+) MS m/e=351 (M+H).

EXAMPLE 31 (SB265609)(±)-5-[1-(3-aminocarbonylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting3-carboxamidepiperidine for benzylamine afforded the title compound as awhite solid in 53% yield. ES (−) MS m/e=390 (M−H).

EXAMPLE 32 (SB266638)(±)-5-[1-(2-Methylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting2-methylpiperidine for benzylamine afforded the title compound as awhite foam in 10% yield. ES (−) MS m/e=361 (M−H).

EXAMPLE 33 (SB266639)(±)-5-[1-(4-Methylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting4-methylpiperidine for benzylamine afforded the title compound as an offwhite solid in 32% yield. ES (−) MS m/e=361 (M−H).

EXAMPLE 34 (SB264732)5-[1-(4-Hydroxypiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting4-hydroxypiperidine for benzylamine afforded the title compound as anoff white solid. ES (+) MS m/e=365 (M+H).

EXAMPLE 35 (SB264733)(±)-5-[1-(2-(2-Hydroxyethyl)piperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting(±)-2-(2-hydroxyethyl)piperidine for benzylamine afforded the titlecompound as an off white solid. ES (−) MS m/e=391 (M−H).

EXAMPLE 36 (SB264734)(±)-5-[1-(3-Hydroxymethylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting(±)-3-hydroxymethylpiperidine for benzylamine afforded the titlecompound as an off white solid. ES (−) MS m/e=377 (M−H).

EXAMPLE 37 (SB264735)5-[1-(4-Phenylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting(±)-3-hydroxymethylpiperidine for benzylamine afforded the titlecompound as an off white solid. ES (−) MS m/e=423 (M−H).

EXAMPLE 38 (SB264736)5-[1-(4-Benzylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting4-benzylpiperidine for benzylamine afforded the title compound as an offwhite solid. ES (−) MS m/e=437 (M−H).

EXAMPLE 39 (SB264863)5-[1-(4-(1-Piperidinyl)piperidinyl)sulfonyl]-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 1b) except substituting4-(1-piperidinyl)piperidine for benzylamine afforded the title compoundas an off white solid. ES (−) MS m/e=430 (M−H).

EXAMPLE 40 (SB263985)5-Benzylaminosulfonyl-N-methyl-3,3-dichloro-2-oxindole a)5-Chlorosulfonyl-N-methyl-3,3-dichloro-2-oxindole

The title compound was prepared according to the procedure of 1a) exceptsubstituting N-methylisatin for isatin. The product was obtained as anorange solid in quantitative yield. ¹H NMR (400 MHz, CDCl₃) d3.37 (s,3H), 7.09 (d, J=10.5 Hz, 1H), 8.16 (d, J=10.5 Hz, 1H), 8.28 (s, 1H).

b) 5-Benzylaminosulfonyl-N-methyl-3,3-dichloro-2-oxindole

To a solution of 5-chlorosulfonyl-N-methyl-3,3-dichloro-2-oxindole (120mg, 382 umol) in methylene chloride (5 mL) was added benzyl amine (50uL, 458 umol) dropwise. After stirring overnight, 3 N hydrochloric acidwas added along with an additional volume of methylene chloride (20 mL).The organic layer was dried over magnesium sulfate, filtered, and silicagel flash chromatography (35 to 55% ethyl acetate/hexanes) yielded thetitle compound. ES (−) MS m/e=383 (M−H).

EXAMPLE 41 (SB263921)N-Methyl-5-(1-piperidinylsulfonyl)-3,3-dichloro-2-oxindole

Prepared according to the procedure of example 41b) except substitutingpiperidine for benzylamine. ¹H NMR (400 MHz, CDCl₃) d1.45 (m, 2H), 1.67(m, 4H), 3.00 (m, 4H), 3.36 (s, 3H), 7.01 (d, J=10.5 Hz, 1H), 7.84 (d,J=10.5 Hz, 1H), 7.99 (s, 1H).

Preparation of Active Caspase 3

Full length Caspase 3 was expressed intracellularly in E.coli withN-terminal hexa His tag. E coli cells were lysed in 10 ml/g of cells oflysis buffer (50 mM Na phosphate pH 7.2, 0.1 M NaCl, 0.1% Tween 20, and10 mM b-mercaptoethanol) using Microfluidics M110Y homogenizer at 10,000psi. After centrifugation, Caspase 3 activity was detected in lysatesupernatant. The supernatant was buffer-exchanged on Sephadex G25 columnequilibrated with 20 mM TrisHCl, 10 % Sucrose, 0.1% CHAPS, 2 mM DTT, pH7.8 (TSCD). Fractions containing Caspase 3 activity was applied to DEAEToyopearl 650 M (Supelco Inc) equilibrated with Buffer TSCD. The columnwas eluted with a linear gradient of 20 mM to 120 mM of Tris Hcl pH 7.8in TSCD. Caspase 3 was eluted in early of the gradient before themajority of impurities eluted. This partially purified Capase 3 was usedfor inhibitor screening. All operations were performed at 4° C. andCaspase activity was measured using substrate, DEVD-AMC, and DynatachFluolite 1000 plate reader.

Caspase 3 Inhibition Assay

Caspase 3 was assayed at 30 degrees C. in 96-well plates using thefluorogenic tetrapeptide substrateN-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartyl-7-amido-4-methylcoumarin(Ac-DEVD-AMC). The assays were conducted at pH 7.5 in a buffered systemcontaining 25 mM Hepes, 10% sucrose, 0.1% CHAPS, and 1-50 uM DTT. Theconcentration of substrate was fixed at 10 uM. Fluorescence of theliberated 7-amino-4-methylcoumarin was continuously monitored at 460 nmfollowing excitation at 360 nm.

Compound Testing

Compounds were tested at a single dose of 50 to 100 uM. Activity wasmonitored as described above over a 30 to 60-minute time periodfollowing the simultaneous addition of substrate and inhibitor to enzymeto initiate the reaction. The progress curves thus generated were fit bycomputer to Eq. 1 in order to assess potency and/or time-dependency:$\begin{matrix}{v = \frac{( {V_{o}( {1 - ^{{- k_{obs}}t}} )} }{k_{obs}}} & (1)\end{matrix}$

Representative compounds of formula (I) have demonstrated positiveinhibitory activity in the above noted assay.

Apoptosis Assay (Jurkat Cells)

Materials: Compounds

Compounds were made as stocks (5-100 mM) in dimethylsulfoxide (DMSO) anddiluted in DMSO to provide final concentrations, with DMSOconcentrations ranging from 0.1-1%.

Preparation of Cells

Jurkat cells were obtained from American Type Culture Collection andgrown in RPMI-1640 media supplemented with 10% fetal bovine serum at37°, 5% CO₂. Cells were seeded in T-flasks at 0.03 to 0.08×10⁶ cells/mland used for experiments at 0.5 to 1.0×10⁶ cells/ml. Other proliferativecells can be used with apoptosis induced by anti-fas, camptothecine,cerimide or TNF.

Apoptosis Assay

A method for measuring apoptosis is to quantitate the amount of brokenDNA fragments using a fluorescent end-labeling method, a system used inthe ApopTag kit from Oncor (Gaithersburg, Md.). In brief, the enzymeterminal deoxynucleotidyl transferase extends the DNA fragments withdigoxigenin-containing nucleotides, which are then dected with anantidigoxigenin antibody carring fluorescein to allow dection byfluorescence (494 nm excitation and 523 nm emission). Propidium iodideis used as counter stain to measure total DNA content. Flow cytometricanalysis was done on Becton-Dickinson (Rutherfor, N.J.) FACScaninstrument using CellQuest software.

METHODS OF TREATMENT

For therapeutic use the compounds of the present invention willgenerally be administered in a standard pharmaceutical compositionobtained by admixture with a pharmaceutical carrier or diluent selectedwith regard to the intended route of administration and standardpharmaceutical practice. For example, they may be administered orally inthe form of tablets containing such excipients as starch or lactose, orin capsule, ovules or lozenges either alone or in admixture withexcipients, or in the form of elixirs or suspensions containingflavouring or colouring agents. They may be injected parenterally, forexample, intravenously, intramuscularly or subcutaneously. Forparenteral administration, they are best used in the form of a sterileaqueous solution which may contain other substances, for example, enoughsalts or glucose to make the solution isotonic with blood. The choice ofform for administration as well as effective dosages will varydepending, inter alia, on the condition being treated. The choice ofmode of administration and dosage is within the skill of the art.

The compounds of the present invention, particularly those noted hereinor their pharmaceutically acceptable salts which are active when givenorally, can be formulated as liquids, for example syrups, suspensions oremulsions, tablets, capsules and lozenges.

A liquid formulation will generally consist of a suspension or solutionof the compound or pharmaceutically acceptable salt in a suitable liquidcarrier(s) for example, ethanol, glycerin, non-aqueous solvent, forexample polyethylene glycol, oils, or water with a suspending agent,preservative, flavouring or colouring agent.

A composition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidformulations. Examples of such carriers include magnesium stearate,starch, lactose, sucrose and cellulose.

A composition in the form of a capsule can be prepared using routineencapsulation procedures. For example, pellets containing the activeingredient can be prepared using standard carriers and then filled intoa hard gelatin capsule; alternatively, a dispersion or suspension can beprepared using any suitable pharmaceutical carrier(s), for exampleaqueous gums, celluloses, silicates or oils and the dispersion orsuspension then filled into a soft gelatin capsule. Preferably thecomposition is in unit dose form such as a tablet or capsule.

Typical parenteral compositions consist of a solution or suspension ofthe compound or pharmaceutically acceptable salt in a sterile aqueouscarrier or parenterally acceptable oil, for example polyethylene glycol,polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.Alternatively, the solution can be lyophilized and then reconstitutedwith a suitable solvent just prior to administration.

A typical suppository formulation comprises a compound or apharmaceutically acceptable salt thereof which is active whenadministered in this way, with a binding and/or lubricating agent suchas polymeric glycols, gelatins or cocoa butter or other low meltingvegetable or synthetic waxes or fats.

The pharmaceutically acceptable compounds of the invention will normallybe administered to a subject in a daily dosage regimen. For a patientthis may be, for example, from about 0.001 to about 100 mg/kg,preferably from about 0.001 to about 10 mg/kg animal body weight. Adaily dose, for a larger mammal is preferably from about 1 mg to about1000 mg, preferably between 1 mg and 500 mg or a pharmaceuticallyacceptable salt thereof, calculated as the free base, the compound beingadministered 1 to 4 times per day. Unit dosage forms may contain fromabout 25 μg to about 500 mg of the compound.

There are many diseases and conditions in which dysregulation ofapoptosis plays an important role. All of these conditions involveundesired, deleterious loss of specific cells with resultingpathological consequences.

Bone remodeling involves the initial resorption by osteoclasts, followedby bone formation by osteoblasts. Recently, there have been a number ofreports of apoptotic events occurring during this process. Apoptoticevents have been observed in both the bone forming and bone resorbingcells in vitro and indeed at the sites of these remodeling units invivo.

Apoptosis has been suggested as one of the possible mechanisms ofosteoclast disappearance from reversal sites between resorption andformation. TGF-β1 induces apoptosis (approx. 30%) in osteoclasts ofmurine bone marrow cultures grown for 6 days in vitro. (Hughes, et al.,J. Bone Min. Res. 9, S138 (1994)). The anti-resorptive bisphosphonates(clodronate, pamidronate or residronate) promote apoptosis in mouseosteoclasts in vitro and in vivo. (Hughes, et al., supra at S347).M-CSF, which has previously been found to be essential for osteoclastformation can suppress apoptosis, suggesting not only that maintenanceof osteoclast populations, but also that formation of thesemultinucleated cells may be determined by apoptosis events. (Fuller, etal., J. Bone Min. Res. 8, S384 (1993); Perkins, et al., J. Bone Min.Res. 8, S390 (1993)). Local injections of IL-1 over the calvaria of miceonce daily for 3 days induces intense and aggressive remodeling.(Wright, et al., J. Bone Min. Res. 9, S174 (1994)). In these studies, 1%of osteoclasts were apoptotic 1 day after treatment, which increased 3days later to 10%. A high percentage (95%) of these apoptoticosteoclasts were at the reversal site. This data suggests that Caspasesare functionally very important in osteoclast apoptosis.

Therefore, one aspect of the present invention is the promotion ofapoptosis in osteoclasts as a novel therapy for inhibiting resorption indiseases of excessive bone loss, such as osteoporosis, using compoundsof Formula (I) as defined herein.

Apoptosis can been induced by low serum in highly differentiated ratosteoblast-like (Ros 17/2.8) cells (Ihbe, et al., (1994) J. Bone Min.Res. 9, S167)). This was associated with a temporal loss of osteoblastphenotype, suggesting that maintenance of lineage specific geneexpression and apoptosis are physiologically linked. Fetal rat calvariaderived osteoblasts grown in vitro undergo apoptosis and this islocalized to areas of nodule formation as indicated by in situend-labeling of fragmented DNA. (Lynch, et al., (1994) J. Bone Min. Res.9, S352). It has been shown that the immediate early genes c-fos andc-jun are expressed prior to apoptosis; c-fos and c-jun-Lac Z transgenicmice show constitutive expression of these transcription factors in veryfew tissues, one of which is bone (Smeyne, et al., (1992) Neuron. 8,13-23; and Morgan, J. (1993) Apoptotic Cell Death: Functions andMechanisms. Cold Spring Harbor October 13-15th). Apoptosis was observedin these animals in the epiphyseal growth plate and chondrogenic zonesas the petula ligament calcifies. Chondrogenic apoptosis has also beenobserved in PTHRP-less mice and these transgenics exhibit abnormalendochondral bone formation (Lee, et al., (1994) J. Bone Min. Res. 9,S159). A very recent paper examined a human osteosarcoma cell line whichundergoes spontaneous apoptosis. Using this cell line, LAP-4, but notICE, could be detected and in vitro apoptosis could be blocked byinhibition or depletion of LAP-4 (Nicholson, et al., (1995) Nature 376,37-43). Thus, apoptosis may play a role in loss of osteoblasts andchondrocytes and inhibition of apoptosis could provide a mechanism toenhance bone formation.

Therefore, another aspect of the present invention is the inhibition ofapoptosis as a novel therapy to enhance bone formation using compoundsof Formula (I) as defined herein.

Osteoarthritits (OA) is a degenerative disease characterized byprogressive erosion of articular cartilage. Chondrocytes are the singlecell-type found in articular cartilage and perturbations in metabolismof these cells may be involved in the pathogenesis of OA. Injury tocartilage initiates a specific reparative response which involves anincrease in the production of proteoglycan and collagen in an attempt toreestablish normal matrix homeostasis. However, with the progress of thedisease, the 3-dimensional collagen network is disrupted and cell deathof chondrocytes occurs in OA lesions (Malemud, et al.: Regulation ofchondrocytes in osteoarthritis. In: Adolphe, M. ed. BiologicalRegulation of Chondrocytes. Boca Raton: CRC Press, 1992, 295-319). Ithas been shown that in OA, chondrocytes adjacent to cartilage defectsexpress high levels of bcl-2 (Erlacher, et al., (1995) J. ofRheumatology, 926-931). This represents an attempt to protectchondrocytes from apoptosis induced by the disease process.

Protection of chondrocytes during early degenerative changes incartilage by inhibition of apoptosis may provide a novel therapeuticapproach to this common disease. Therefore, another aspect of thepresent invention is the inhibition of apoptosis as a novel therapy totreat osteoarthritis, using compounds of Formula (I) as defined herein.

Recent evidence shows that chronic, degenerative conditions of the liverare linked to hepatocellular apoptosis. These conditions includechemical-, infectious- and immune/inflammatory-induced hepatocellulardegeneration. Apoptosis of liver cells has been observed in liverdegenerative states induced by a variety of chemical agents, includingacetaminophen (Ray, et al., (1993) FASEB. J. 7, 453-463), cocaine(Cascales, et al., (1994) Hepatology 20, 992-1001) and ethanol (Baroni,etal., (1994) J. Hepatol. 20, 508-513). Infectious agents and theirchemical components that have been shown to induce apoptosis includehepatitis ((Hiramatsu, et al., (1994) Hepatology 19, 1354-1359; Mita, etal., (1994) Biochem. Biophys. Res. Commun. 204, 468-474)), tumornecrosis factor and endotoxin. (Leist, et al., (1995) J. Immunol. 154,1307-1316; and Decker, K. (1993) Gastroenterology 28(S4), 20-25).Stimulation of immune/inflammatory responses by mechanisms such asallograft transplantation and hypoxia followed by reperfusion have beenshown to induce apoptosis of hepatocytes (Krams, et al., (1995)Transplant. Proc. 27, 466-467). Together, this evidence supports thathepatocellular apoptosis is central to degenerative liver diseases.

Therefore, another aspect of the present invention is the inhibition ofapoptosis as a novel therapy to treat degenerative liver diseases, usingcompounds of Formula (I) as defined herein.

Apoptosis is recognized as a fundamental process within the immunesystem where cell death shapes the immune system and effects immunefunctions. Apoptosis also is implicated in viral diseases (e.g AIDS).Recent reports indicate that HIV infection may produce an excess ofapoptosis, contributing to the loss of CD4⁺ T cells. Of additionalinterest is the observation that APO-1/Fas shares sequence homology withHIV-1 gp120.

Therefore, another aspect of the present invention is the inhibition ofapoptosis as a novel therapy to treat viral diseases, using compounds ofFormula (I) as defined herein.

Additional therapeutic directions and other indications in whichinhibition of apoptotic cysteine proteases is of therapeutic utility,along with relevant citations in support of the involvement forapoptosis in each indication, are presented below in Table 1.

Indication Citations Ischemia/reperfusion Barr et al., (1994)BioTechnology 12, 487- 493; Thompson, C. B. (1995) Science 267,1456-1462 Stroke Barr et al supra; and Thompson, C., supra Polycystickidney disease Barr et al., supra; and Mondain, et al., (1995) ORL J.Otorhinolaryngol. Relat. Spec. 57, 28-32 Glomerulo-nephritis Barr etal., supra Osteoporosis Lynch et al., (1994) J. Bone Min. Res. 9, S352;Nicholson et al., (1995) Nature 376, 37-43 Erythropoiesis/ Thompson, C.,supra; Koury et al., (1990) Aplastic anemia Science 248, 378-381 Chronicliver degeneration Thompson, C., supra; Mountz et al., 1994) ArthritisRheum. 37, 1415-1420; Goldin et al., (1993) Am. J. Pathol. 171, 73- 76T-cell death Thompson, C., supra; Ameison et al., (1995) Trends CellBiol. 5, 27-32 Osteoarthritis-chondrocytes Ishizaki et al., (1994) J.Cell Biol. 126, 1069-1077; Blanco et al., (1995) Am. J. Pathol. 146,75-85 Male pattern baldness Mondain et al., supra; Seiberg et al.,(1995) J. Invest. Dermatol. 104, 78-82; Tamada et al., (1994) Br. J.Dermatol. 131, 521-524 Alzheimer's disease Savill, J., (1994) Eur. J.Clin. Invest. 24, 715-723; Su et al., (1994) Neuroreport 5, 2529-2533;Johnson, E., (1994) Neurobiol. Aging 15 Suppl. 2, S187-S189 Parkinson'sdisease Savill, J., supra; Thompson. C., supra Type I diabetes Barr etal., supra

The IL-1 and TNF inhibiting effects of compounds of the presentinvention are determined by the following in vitro assays:

Interleukin-1 (IL-1)

Human peripheral blood monocytes are isolated and purified from eitherfresh blood preparations from volunteer donors, or from blood bank buffycoats, according to the procedure of Colotta et al. J Immunol, 132, 936(1984). These monocytes (1×10⁶) are plated in 24-well plates at aconcentration of 1-2 million/ml per well. The cells are allowed toadhere for 2 hours, after which time non-adherent cells are removed bygentle washing. Test compounds are then added to the cells for about 1hour before the addition of lipopolysaccharide (50 ng/ml), and thecultures are incubated at 37° C. for an additional 24 hours. At the endof this period, culture super-natants are removed and clarified of cellsand all debris. Culture supernatants are then immediately assayed forIL-1 biological activity, either by the method of Simon et al., J.Immunol. Methods, 84, 85, (1985) (based on ability of IL-1 to stimulatea Interleukin 2 producing cell line (EL4) to secrete IL-2, in concertwith A23187 ionophore) or the method of Lee et al., J. ImmunoTherapy, 6(1), 1-12 (1990) (ELISA assay).

Tumour Necrosis Factor (TNF)

Human peripheral blood monocytes are isolated and purified from eitherblood bank buffy coats or platelet pheresis residues, according to theprocedure of Colotta, R. et al., J Immunol, 132(2), 936 (1984). Themonocytes are plated at a density of 1×10⁶ cells/ml medium/well in24-well multi-dishes. The cells are allowed to adhere for 1 hour afterwhich time the supernatant is aspirated and fresh medium (1 ml,RPMI-1640, Whitaker Biomedical Products, Whitaker, Calif.) containing 1%fetal calf serum plus penicillin and streptomycin (10 units/ml) added.The cells are incubated for 45 minutes in the presence or absence of atest compound at 1 nM-10 mM dose ranges (compounds are solubilized indimethyl sulfoxide/ethanol, such that the final solvent concentration inthe culture medium is 0.5% dimethyl sulfoxide/0.5% ethanol). Bacteriallipopoly-saccharide (E. coli 055:B5 [LPS] from Sigma Chemicals Co.) isthen added (100 ng/ml in 10 ml phosphate buffered saline) and culturesincubated for 16-18 hours at 37° C. in a 5% CO₂ incubator. At the end ofthe incubation period, culture supernatants are removed from the cells,centrifuged at 3000 rpm to remove cell debris. The supernatant is thenassayed for TNF activity using either a radio-immuno or an ELISA assay,as described in WO 92/10190 and by Becker et al., J Immunol, 1991, 147,4307.

The above description fully discloses the invention including preferredembodiments thereof. Modifications and improvements of the embodimentsspecifically disclosed herein are within the scope of the followingclaims. Without further elaboration, it is believed that one skilled inthe are can, using the preceding description, utilize the presentinvention to its fullest extent. Therefore the Examples herein are to beconstrued as merely illustrative and not a limitation of the scope ofthe present invention in any way. The embodiments of the invention inwhich an exclusive property or privilege is claimed are defined asfollows.

What is claimed is:
 1. A compound of formula

wherein R₁ is hydrogen, or C₁₋₄ alkyl; R₂ is C₁₋₁₀ alkyl, optionallysubstituted arylC₁₋₄alkyl, optionally substituted heteroaryl C₁₋₄ alkyl,optionally substituted C₃₋₇ cycloalkyl, or R₁ and R₂ together with thenitrogen to which they are attached from a 3 to 10 membered ring whichoptionally contains an aditional heteroatom selected from oxygen,nitrogen or sulfur; R₃ and R₄ are C₁₋₆alkyl, hydrogen, nitro, or halogenand R₅ is C₁₋₆alkyl, hydrogen, arylalkyl or heteroarylalkyl.
 2. Acompound according to claim 1 wherein R₅ is substituted benzyl.
 3. Acompond according to claim 1 wherein R₁ is hydrogen or methyl.
 4. Acompound according to claim 1 wherein R₁ and R₂ are joined to form afive membered nitrogen containing ring.
 5. The compound according toclaim 1 which is 5-Chlorosulfonyl-3,3-dichloro-2-oxindole5-Benzylaminosulfonyl-3,3-dichloro-2-oxindole5-[N-(1-Methyl-3-phenylpropylamino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(N-Benzyl-2-cyanoethylamino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(2-(3-Pyridyl)ethylamino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(2-Furfurylamino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(2-Isopropoxyethylamino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(2-Methoxyethylamino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(2-Tetrahydrofurfurylamino)sulfonyl]-3,3-dichloro-2-oxindole(−)-5-[N-(cis-Myrtanylamino)sulfonyl]-3,3-dichloro-2-oxindole5-[(1-Benzylpiperidinyl-4-amino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(2-Indanamino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(Cyclopropylmethylamino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(1,5-Dimethylhexylamino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(N-Methylbenzylamino)sulfonyl]-3,3-dichloro-2-oxindole(±)-5-[N-(3-(N-Acetyl-N-methylamino)pyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole5-[2-(1,2,3,4-Tetrahydroisoquinolino)sulfonyl]-3,3-dichloro-2-oxindole(±)-5-[1-(Decahydroisoquinolino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(N-Methyl-2-cyanoethylamino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(N-Methylcyanomethylamino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(Pyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(N-Methylphenethylamino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(Azacyclooctane)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(3-Azabicyclo[3.2.2]nonane)sulfonyl]-3,3-dichloro-2-oxindole5-[1-(2-Ethoxycarbonylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(Morpholino)sulfonyl]-3,3-dichloro-2-oxindole(S)-(+)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(N-Methyl-2-(4-pyridinyl)ethylamino)sulfonyl]-3,3-dichloro-2-oxindole5-[N-(N-Methyl-2-hydroxyethylamino)sulfonyl]-3,3-dichloro-2-oxindole(S)-(+)-5-[N-(2-Hydroxymethylpyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole(±)-5-[1-(3-Hydroxypyrrolidinyl)sulfonyl]-3,3-dichloro-2-oxindole(±)-5-[1-(3-aminocarbonylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole(±)-5-[1-(2-Methylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole(±)-5-[1-(4-Methylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole5-[1-(4-Hydroxypiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole(±)-5-[1-(2-(2-Hydroxyethyl)piperidinyl)sulfonyl]-3,3-dichloro-2-oxindole(±)-5-[1-(3-Hydroxymethylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole5-[1-(4-Phenylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole5-[1-(4-Benzylpiperidinyl)sulfonyl]-3,3-dichloro-2-oxindole5-[1-(4-(1-Piperidinyl)piperidinyl)sulfonyl]-3,3-dichloro-2-oxindole5-Benzylaminosulfonyl-N-methyl-3,3-dichloro-2-oxindole5-Chlorosulfonyl-N-methyl-3,3-dichloro-2-oxindole5-Benzylaminosulfonyl-N-methyl-3,3-dichloro-2-oxindoleN-Methyl-5-(1-piperidinylsulfonyl)-3,3-dichloro-2-oxindole.
 6. Apharmaceutical composition comprising a compound according to claim 1and a pharmaceutically acceptable carrier or diluent.
 7. A method ofblocking excess or inappropriate apoptosis in a mammal in need of suchtreatment which method comprises administering to said mammal or humanan effective amount of a compound of Formula (I), according to claim 1or a pharmaceutically acceptable salt thereof.
 8. The method accordingto claim 7 wherein the excessive or inappropriate apoptosis occurs inAlzheimer disease.
 9. The method according to claim 7 wherein theexcessive or inappropriate apoptosis occurs in viral infections.
 10. Themethod according to claim 7 wherein the excessive or inappropriateapoptosis occurs during infarction or reperfusion injury.
 11. The methodaccording to claim 7 wherein the excessive or inappropriate apoptosisoccurs during ischemia.
 12. The method according to claim 7 wherein theexcessive or inappropriate apoptosis results in excessive bone loss. 13.The method according to claim 7 wherein the excessive or inappropriateapoptosis results in the disease of osteoarthritis.
 14. The methodaccording to claim 7 wherein the excessive or inappropriate apoptosisresults in hepatocellular degeneration.
 15. A method for the treatmentof diseases or disorders associated with excessive IL-1β convertaseactivity, in a mammal in need thereof, which method comprisesadministering to said mammal an effective amount of a compound ofFormula (I), according to claim 1 or a pharmaceutically acceptable saltthereof.
 16. A method of blocking or decreasing the production of IL-1βand/or TNF, in a mammal in need of such treatment, which methodcomprises administering to said mammal an effective amount of a compoundof Formula (I) according to claim 1, or a pharmaceutically acceptablesalt thereof.
 17. A method for inhibiting the production of caspasethree and seven in a mammal in need of such treatment, which methodcomprises administering to said mammal an effective amount of a compoundof Formula (I) according to claim 1, or a pharmaceutically acceptablesalt thereof.